CN105980855A

CN105980855A - High-throughput and highly multiplexed imaging with programmable nucleic acid probes

Info

Publication number: CN105980855A
Application number: CN201580006969.2A
Authority: CN
Inventors: 尹鹏; S·阿加斯蒂; 陈曦; R·容曼
Original assignee: Harvard College
Current assignee: Harvard College
Priority date: 2014-03-11
Filing date: 2015-03-11
Publication date: 2016-09-28
Anticipated expiration: 2035-03-11
Also published as: US9944972B2; EP4030166A1; AU2015229406B2; WO2015138653A1; EP3117214A4; AU2020239730B2; IL246962B; JP2017509872A; BR112016018314A2; RU2016139490A; US10190151B2; CN110004208A; US20170137864A1; EP4245859A2; KR102313982B1; JP2020193988A; KR20210127799A; CN105980855B; AU2015229406A1; CA2932943C

Abstract

The present invention provides, inter alia, methods and compositions for imaging, at high spatial resolution, targets of interest.

Description

Utilize high flux and the imaging of height multiplexing of nucleic probe able to programme

Related application

This application claims the rights and interests of the U.S. Provisional Application No. 61/951,461 submitted on March 11st, 2014, its Full content is incorporated herein by reference.

Invention field

Present invention relates in general to the field of detection and analyte quantification (such as, target).

Background of invention

Fluorescence microscopy is the powerful of a kind of molecule for exploring in such as biosystem. However, it is possible to be distinguished ground and the number (i.e. multiplexing ability) of the most visual different plant species Limited by the spectra overlapping between fluorogen.

Summary of the invention

Invention particularly provides for detecting, imaging and/or quantify target interested (such as, Biomolecule) method and composition.Some in method include that (1) makes one to need provided herein The sample (such as, a doubtful sample containing one or more targets interested) analyzed is with special Property combine the part binding partners of a given target (each part be) contact of these targets, its In each moiety to a nucleic acid (referred to here as butt chain) and wherein have the most homospecific Binding partners is coupled to different butt chain, and (2) optionally remove unconjugated binding partners, and (3) make This sample and labeled (such as, fluorescently-labeled) nucleic acid contact, and these nucleic acid have and are complementary to also And (this type of labeling nucleic acid is referred to here as labeled one-tenth to be therefore specific to the nucleotide sequence of a butt chain As chain), (4) optionally remove unconjugated imaging chain, (5) by sample imaging in whole or in part to examine Surveying position and the number of combined imaging chain, (6) quencher is from the signal of the labeled imaging chain of sample (such as, by bleaching, including photobleaching), and (7) repetition step (3)-(6), use one every time The individual imaging for other imaging chains whole used in this method with unique nucleotide sequence Chain.

Imaging chain can be by labelling in the same manner, including being carried out fluorescent labeling in the same manner.In other embodiments In, the imaging chain with an identical sequence can be by labelling in the same manner.First method is probably more square Just, because it needs single excitation wavelength and detector.

In such a way, can detect, imaging and/or quantify two or more in a sample Individual target, regardless of they positions in this sample, including no matter they positions in the sample are No closely, if so that observe from the two or the signal of more target simultaneously, being can not Difference.Therefore, the distance between two or more targets is smaller than the imaging system for detecting target The resolution distance of system, and still uses method provided herein, can by two or more targets that This differentiates, and thus promotes the more accurate and sane detection to this type of target and quantization.In certain situation Under, this resolution distance can be about 50nm, as an example.

Should be appreciated that before carrying out the method, " the target content " of a sample is probably known or bosom That doubt or unknown and unsuspected.Contact sample binding partners can in conjunction with or not can be coupled to this On sample, this depends on whether target exists, and (such as, in the presence of target, this binding partners can be tied Close on this sample).Contact sample imaging chain can in conjunction with or not can be coupled on this sample, this depends on Whether have in target that (such as, in the presence of target, this imaging chain can be attached on target in conjunction with one Corresponding butt chain)." it is attached on sample " mean that binding partners or imaging chain are incorporated into it right On the target answered or butt chain.

The protein that binding partners can be naturally-occurring, such as antibody or antibody fragment.Join in combination In the case of even body is a kind of antibody or antibody fragment, butt chain can be coupled to it a constant region On.Binding partners can be the antibody of a kind of such as monoclonal antibody, or it can be that antigen combines Antibody fragment, such as from the Fab of monoclonal antibody.In certain embodiments, in conjunction with joining Even body is a kind of receptor.

Binding partners can be connected to butt chain by a transition joint.In certain embodiments, one Individual transition joint comprises biotin and/or Succ-PEG-DSPE.

Imaging chain can be carried out fluorescent labeling (that is, they being coupled to a fluorogen).It is coupled to The fluorogen of the imaging chain with different IPs nucleotide sequence can be mutually the same, or they can have Overlapping with other fluorogens or nonoverlapping transmitting scattergram.Fluorescently-labeled imaging chain can include at least one Individual fluorogen.

In some cases, the fluorescently-labeled imaging nucleic acid of such as imaging chain can include 1,2,3 or more Multiple fluorogens.

This sample can be cell, cell mass or from cell or the cell lysate of cell mass.Should Target can be protein.

It will be appreciated that arrive, the present invention provides a kind of method for detecting analyte, and the method is to pass through Analyte is attached on the binding partners of its correspondence and sequentially determines depositing of this type of binding partners , by repeatedly combine, detect and quencher (such as, bleaching, such as photobleaching) optionally by The imaging chain of labelling (such as, in the same manner fluorescent labeling) in the same manner.

Therefore, present disclosure provides a kind of method, makes one to need for one or more targets including (1) The existence sample that carries out testing contact with one or more target specificity binding partners, the most each Target specificity binding partners is connected to a butt chain, and wherein has the most homospecific target Specific binding partner is connected to different butt chain, and (2) optionally remove unconjugated target specificity knot Closing gametophyte, (3) make this sample and have the labeled imaging of the nucleotide sequence being complementary to a butt chain Chain contact, (4) optionally remove unconjugated imaging chain, (5) by this sample imaging in case detection combine The position of labeled imaging chain and number, (6) quencher from the signal of labeled imaging chain combined, and (7) repeat step (3)-(6), use one to have for all other labeled imaging chains every time The labeled imaging chain of unique nucleotide sequence.

In certain embodiments, this sample is combined with more than one target specificity in step (1) and joins Even body contact.

In certain embodiments, this target specificity binding partners is a kind of antibody or antibody fragment.

In certain embodiments, these labeled imaging chains are by labelling in the same manner.In certain embodiments, These labeled imaging chains each include a different labelling.In certain embodiments, these are labeled Imaging chain is fluorescently-labeled imaging chain.

In certain embodiments, these one or more targets are protein.In certain embodiments, this sample Product are cell, cell lysate or Tissue Lysates.

In certain embodiments, use copolymerization burnt or epifluorescence microscope inspection art should in step (5) Sample imaging.

In certain embodiments, the quencher signal in step (6) includes photobleaching.

Present disclosure further provides for being attached to more than one target identification division (such as target a kind of comprising Specific binding partner) on the compositions of sample, each target identification division is attached to a docking On nucleic acid (such as butt chain), and at least one is attached to labeled imaging core with docking nucleic acid stability In acid (such as imaging chain).

Present disclosure further provides for a kind of compositions, and said composition comprises that to be attached to more than one target special A sample on specific binding partner, on each binding partner binds to butt chain；And At least one is stably coupled to the butt chain on labeled imaging chain.

Present disclosure further provides for a kind of method, makes one to need for one or more targets including (1) Existence carry out the sample tested (such as target specificity be combined and join with one or more target identification divisions Even body) contact, the most each target identification division is connected to docking nucleic acid (such as butt chain), And wherein having the most homospecific target identification division and be connected to different docking nucleic acid, (2) are optionally gone Except unconjugated target identification division, (3) make this sample and are complementary to the nucleotide docking nucleic acid with having Labeled imaging nucleic acid (the such as imaging chain) contact of sequence, (4) optionally remove unconjugated labeled Imaging nucleic acid, (5) by sample imaging so that the position of labeled imaging nucleic acid that combines of detection and number, (6) removed from docking nucleic acid by change temperature and/or the combining labeled imaging nucleic acid of buffer condition, And (7) repeat step (3)-(6), use one to come relative to all other labeled imaging nucleic acid every time Say the labeled imaging nucleic acid with unique nucleotide sequence.Under such conditions, this imaging nucleic acid is spontaneous Ground dissociates from docking nucleic acid.

Present disclosure further provides for a kind of method, makes one to need for one or more targets including (1) The existence sample that carries out testing contact with one or more target specificity binding partners, the most each Target specificity binding partners is connected to a docking nucleic acid, and wherein has the most homospecific target Mark specific binding partner is connected to different docking nucleic acid, and it is special that (2) optionally remove unconjugated target Property binding partners, (3) make this sample with have be complementary to a nucleotide sequence docking nucleic acid through mark Note imaging nucleic acid contact, (4) optionally remove unconjugated labeled imaging nucleic acid, and (5) are by sample imaging So that the position of the labeled imaging nucleic acid that detection combines and number, (6) are by changing temperature and/or buffering The combining labeled imaging nucleic acid of condition is removed from docking nucleic acid, and (7) repeat step (3)-(6), Use a warp for all other labeled imaging nucleic acid with unique nucleotide sequence every time It is marked as nucleic acid.Under such conditions, this imaging nucleic acid spontaneously dissociates from docking nucleic acid.

In certain embodiments, by reducing salinity, adding denaturant or improve temperature by labeled Imaging nucleic acid is removed from docking nucleic acid.In certain embodiments, this salt is Mg++.In some embodiments In, this denaturant is Methanamide, carbamide or DMSO.

Present disclosure further provides for a kind of method, makes one to need for one or more targets including (1) Existence carry out the sample tested (such as target specificity be combined and join with one or more target identification divisions Even body) contact, the most each target identification division is connected to docking nucleic acid (such as butt chain), And wherein having the most homospecific target identification division and be connected to different docking nucleic acid, (2) are optionally gone Except unconjugated target identification division, (3) make this sample and are complementary to the nucleotide docking nucleic acid with having Labeled imaging nucleic acid (the such as imaging chain) contact of sequence, (4) optionally remove unconjugated labeled Imaging nucleic acid, (5) by sample imaging so that the position of labeled imaging nucleic acid that combines of detection and number, (6) combining labeled imaging nucleic acid is removed from docking nucleic acid, and (7) repeat step (3)-(6), Use a warp for all other labeled imaging nucleic acid with unique nucleotide sequence every time It is marked as nucleic acid.

Present disclosure further provides for a kind of method, makes one to need for one or more targets including (1) The existence sample that carries out testing contact with one or more target specificity binding partners, the most each Target specificity binding partners is connected to a docking nucleic acid, and wherein has the most homospecific target Mark specific binding partner is connected to different docking nucleic acid, and it is special that (2) optionally remove unconjugated target Property binding partners, (3) make this sample with have be complementary to a nucleotide sequence docking nucleic acid through mark Note imaging nucleic acid contact, (4) optionally remove unconjugated labeled imaging nucleic acid, and (5) are by sample imaging So as the position of the labeled imaging nucleic acid that detection combines and number, (6) combining labeled imaging nucleic acid Remove from docking nucleic acid, and (7) repeat step (3)-(6), use every time one relative to all other There is for labeled imaging nucleic acid the labeled imaging nucleic acid of unique nucleotide sequence.

In certain embodiments, in step (6), in the presence of a competition nucleic acid, do not pass through chain Replace and labeled imaging nucleic acid is removed from docking nucleic acid.

In certain embodiments, in step (6), split by chemical method, photochemical method or enzymatic method Solution, labeled imaging nucleic acid of modifying or degrade, remove labeled imaging nucleic acid from docking nucleic acid.

In certain embodiments, when labeled imaging nucleic acid is attached on the docking nucleic acid of its correspondence, Strand district is there is not on imaging nucleic acid or docking nucleic acid.In certain embodiments, this docking nucleic acid does not has Foothold sequence (toehold sequence).In certain embodiments, this imaging nucleic acid does not have and bases oneself upon Point sequence.

In certain embodiments, this labeled imaging nucleic acid is not self-quenching.

Present disclosure further provides for a kind of method, makes one to need for one or more targets including (1) Existence carry out the sample tested (such as target specificity be combined and join with one or more target identification divisions Even body) contact, the most each target identification division is connected to docking nucleic acid (such as butt chain), And wherein having the most homospecific target identification division and be connected to different docking nucleic acid, (2) are optionally gone Except unconjugated target identification division, (3) make this sample and are complementary to the nucleotide docking nucleic acid with having Labeled imaging nucleic acid (the such as imaging chain) contact of sequence, (4) optionally remove unconjugated labeled Imaging nucleic acid, (5) by sample imaging so that the position of labeled imaging nucleic acid that combines of detection and number, (6) not exclusively remove imaging nucleic acid inactivate combination by removing or modify their signal emission part Labeled imaging nucleic acid, and (7) repeat step (3)-(6), use every time one relative to all other There is for labeled imaging nucleic acid the labeled imaging nucleic acid of unique nucleotide sequence.

Present disclosure further provides for a kind of method, makes one to need for one or more targets including (1) The existence sample that carries out testing contact with one or more target specificity binding partners, the most each Target specificity binding partners is connected to a docking nucleic acid, and wherein has the most homospecific target Mark specific binding partner is connected to different docking nucleic acid, and it is special that (2) optionally remove unconjugated target Property binding partners, (3) make this sample with have be complementary to a nucleotide sequence docking nucleic acid through mark Note imaging nucleic acid contact, (4) optionally remove unconjugated labeled imaging nucleic acid, and (5) are by sample imaging So that the position of the labeled imaging nucleic acid that detection combines and number, (6) are by removing or modify their letter Number emitting portion and not exclusively remove imaging nucleic acid to inactivate the labeled imaging nucleic acid of combination, and (7) Repeat step (3)-(6), use one to have solely for all other labeled imaging nucleic acid every time The labeled imaging nucleic acid of special nucleotide sequence.

Different embodiments are equally applicable to preceding method.These embodiments are as follows:

In certain embodiments, this sample is combined with more than one target specificity in step (1) and joins Even body contact.In certain embodiments, this target specificity binding partners is a kind of antibody or antibody sheet Section.In certain embodiments, this target specificity binding partners is natural or the part of through engineering approaches, little Molecule, fit, peptide or oligonucleotide.

In certain embodiments, these labeled imaging nucleic acid are by labelling in the same manner.In some embodiments In, these labeled imaging nucleic acid each include a different labelling.In certain embodiments, these Labeled imaging nucleic acid is fluorescently-labeled imaging nucleic acid.

In certain embodiments, this unconjugated docking nucleic acid is partially double stranded.In some embodiments In, this unconjugated imaging nucleic acid is partially double stranded.

In certain embodiments, this imaging nucleic acid is a molecular beacon or includes a hairpin secondary knot Structure.In certain embodiments, this imaging nucleic acid is a molecular beacon or includes sending out of a self-quenching Folder secondary structure.In certain embodiments, this imaging nucleic acid is one and half duplexs (hemiduplex). In certain embodiments, this half duplex is self-quenching.In certain embodiments, this imaging nucleic acid leads to Cross a dendritic structure or a paradigmatic structure is attached in multiple signal emission part.This imaging nucleic acid It can be straight or branched.

In certain embodiments, this docking nucleic acid includes a hairpin secondary structures.

These and other embodiments will illustrate in more detail at this.

Accompanying drawing is sketched

Fig. 1 is a kind of high flux and the most scalable multiplexing imaging side method provided in present disclosure The schematic diagram of an embodiment.By cell imaging and then at the one-tenth of round subsequently after probe hybridizes As carrying out photobleaching before.

Fig. 2 is a kind of high flux based on buffer exchange and the most scalable multiplexing imaging side method The schematic diagram of an embodiment, this buffer exchange uses the solution with slight Vertic features, such as Salinity, the concentration of forma of increase or the higher temperature reduced.

Fig. 3 is by using the method provided in present disclosure removal imaging chain to inactivate one of this imaging chain The schematic diagram of embodiment.

Fig. 4 is not removed the nucleic acid moiety of imaging chain to inactivate one of this imaging chain by inactivation fluorogen The schematic diagram of embodiment.

Fig. 5 is the schematic diagram of an embodiment of the self-quenching imaging chain of a molecular beacon sample.

Fig. 6 is the schematic diagram of an embodiment of one and half duplex self-quenching imaging chains.

Fig. 7 is the schematic diagram of an embodiment of a non-strand butt chain.

Fig. 8 is the schematic diagram of an embodiment of an imaging chain, and multiple signal emission part raised by this imaging chain Divide and be copied to butt chain.

Fig. 9 is the schematic diagram of an embodiment of a non-strand imaging chain.

Figure 10 is 10 the different oligonucleotide being shown under 3 kinds of different conditions the reverse complemental chain with correspondence The figure of prediction dissociation constant.Sequence a1:5 '-CATCTAAAGCC-3 ' (SEQ ID NO:1)；Sequence Row a2:5 '-GAATTTCCTCG-3 ' (SEQ ID NO:2)；Sequence a3:5 '- GTTTAATTGCG-3’(SEQ ID NO:3)；Sequence a4:5 '-ACAATTCTTCG-3 ' (SEQ ID NO:4)；Sequence a5:5 '-TTTCTTGCTTC-3 ' (SEQ ID NO:5)；Sequence A6:5 '-GCATTGTTACT-3 ' (SEQ ID NO:6)；Sequence a7:5 '-ATATACAAGCG- 3’(SEQ ID NO:7)；Sequence a8:5 '-GCTGTCTATTG-3 ' (SEQ ID NO:8)；Sequence Row a9:5 '-TCTTTATGCTG-3 ' (SEQ ID NO:9)；Sequence a10:5 '- CAATCTCATCC-3’(SEQ ID NO:10)。

Invention description

Invention particularly provides such as, using image probe based on nucleic acid (such as, based on DNA Image probe) cellular environment in for multiplexing compositions and the method for fluorescence imaging.Carry at this The method of confession is to be based in part on nucleic acid butt chain and the programmability of imaging chain.It is to say, such as, Butt chain and imaging chain can be designed, during so that they are bonded to each other certain under certain conditions Between.This programmability permission imaging chain stable bond is to butt chain, as provided here.Generally, What method related to identifying in a specific sample (such as, biological sample) provided herein is one or more Target (such as, one or more biomolecule of such as protein or nucleic acid).In some cases, Whether one or more targets are present in sample is unknown.Therefore, the method for present disclosure can be used for really It is scheduled in a doubtful sample containing one or more targets and whether there are this one or more targets.? Provided herein in any one of aspect and embodiment, sample can containing or can doubtful containing one or Multiple targets.

Therefore, the present invention is provided to carry out high flux based on nucleic acid able to programme (such as, DNA) probe And height multiplexing imaging and the method for analyte/target detection.These methods depend on one and adopt With the order formation method of orthogonal imaging chain, these orthogonal imaging chains can be stably attached to one and fix Complementary pair chain link (Fig. 1) on binding partners (such as antibody).With the hybridization of imaging chain and After imaging, carry out a quenching step (a such as photobleaching step) so as to eliminate and/or reduce from Hybridization (in conjunction with) the fluorescence of imaging chain.

In another embodiment, these methods utilize the relatively weak binding between butt chain and imaging chain so that Remove signal.Such as, hybridization conditions can change, and so makes formation between butt chain or imaging chain The fusing point of duplex is slightly above room temperature (such as, 25 DEG C) or imaging temperature.It is marked as described above Step (i.e., wherein imaging chain combination is to the step on the butt chain of its correspondence) and image-forming step.As One example, after first target is carried out imaging, makes this sample stand a Denaturing.Can make By one, there is the most relatively low salt concn, there is or increase the concentration of denaturant (such as Methanamide), or The solution of the temperature increased, provides this Denaturing (Fig. 2) in buffer exchange step.This sample can To be alternatively or additionally exposed to the temperature of increase.Above-mentioned being increased or decreased is relative at labelling Condition (that is, when in imaging chain combination to butt chain) present in step.Feelings in buffer exchange Under condition, this sample washable, this buffer exchange repeatable, can again wash this sample, and then Add next one imaging chain to this sample.

For multiplexing, such as, going out for the photobleaching of quencher signal or additive method or imaging chain Use the different banks of orthogonal imaging chain sequentially to same sample after each step lived or remove, with The unlimited number of target of imaging the most potentially.It is different from traditional formation method and multiplexes by color Spectra overlapping between passage limits, and method is only by (butt chain or alternately imaging provided herein Chain) restriction of possible orthogonal nucleotides sequence column number.Due to can easily design larger amount of just Handing over nucleotide sequence, the method has only the most scalable by use single fluorogen just to reach Multiplexing ability.The method can easily arrange with standard microscope inspection art (such as, copolymerization burnt or Epifluorescence microscope inspection art) integrate, thus allow to analyze this sample with high throughput.

These methods have being suitable in such as high flux screening measures (such as drug screening mensuration) Property.This formation method allows using standard copolymerization burnt or while epifluorescence microscope inspection art imaging, The cell mass (～1,000-10,000) big with a kind of super multiplexing format analysis or tissue sample.With one Plant high-throughout mode and from same sample, screen a large amount of target (such as protein) by offer about new drug Or the information of regulator, cell heterogeneity information is provided simultaneously.There is high flux and the one-tenth of super multiplexing As the Large-scale Screening of the tissue sample of ability will be applicable to pathological analysis, such as, hospital or other During service provider is arranged.

Method can be also used for identifying that a single target is (such as, such as, a kind of provided herein Specific protein) absolute magnitude, or a single target is for other targets one or more Amount.

It addition, method can be used for identifying that a target is in a sample or relative to sample provided herein In the position of other targets.

Therefore, present disclosure provides a kind of method, including (1) make a sample simultaneously with multiple sequence marks Target identification division contact, (2) introduce imaging nucleic acid (such as imaging chain), these imaging nucleic acid pass through Docking nucleic acid (such as butt chain) in the target identification division of complementarity recognition sequence labelling is sub- Group, imaging nucleic acid or the quencher signal from imaging nucleic acid is removed or inactivated in (3), and (4) are at least Once repeat step (2) and optionally step (3) in case imaging and detect one or more other right Connect nucleic acid.

The method optionally includes coming labelling multiple target identification part with docking nucleic acid (such as butt chain) Point so that the target identification division of formation sequence labelling.

Present disclosure provides a kind of method, makes an existence needed for one or more targets including (1) The sample carrying out testing contacts with one or more target specificity binding partners, and the most each target is special Specific binding partner is connected to a butt chain, and wherein has the most homospecific target specificity Binding partners is connected to different butt chain, and (2) are optionally removed unconjugated target specificity and combined spouse Body, (3) make this sample and the labeled imaging with the nucleotide sequence being complementary to a butt chain links Touch, (4) optionally remove unconjugated imaging chain, (5) by this sample imaging in case detection combine through mark The position of note imaging chain and number, (6) quencher is from the signal of the labeled imaging chain combined, and (7) Repeat step (3)-(6), use one to have uniqueness for all other labeled imaging chains every time The labeled imaging chain of nucleotide sequence.

Step (3)-(6) may be repeated one or more times.Such as, repeatable 1-10 time or more of step (3)-(6) Repeatedly.In certain embodiments, step (3)-(6) repeat 1,2,3,4,5,6,7,8,9 or 10 Secondary.

Present disclosure further provides for a kind of method, makes one to need for one or more targets including (1) Existence carry out the sample tested (such as target specificity be combined and join with one or more target identification divisions Even body) contact, the most each target identification division is connected to a docking nucleic acid, and wherein has not Homospecific target identification division is connected to different docking nucleic acid, and (2) optionally remove unconjugated target Identification division, (3) make this sample with have be complementary to one docking nucleic acid nucleotide sequence labeled become As nucleic acid (such as imaging chain) contact, (4) optionally remove unconjugated labeled imaging nucleic acid, (5) By sample imaging so that the position of the labeled imaging nucleic acid of detection combination and number, (6) are combining through mark Note imaging nucleic acid is removed from docking nucleic acid, and (7) repeat step (3)-(6), uses one relatively every time There is for all other labeled imaging nucleic acid the labeled imaging nucleic acid of unique nucleotide sequence.

In certain embodiments, method includes following steps provided herein: use a kind of except chain Method outside displacement is removed and is attached to dock the imaging nucleic acid on nucleic acid (such as butt chain) (such as Imaging chain).

In certain embodiments, method includes that a removal is attached to dock nucleic acid (such as provided herein Butt chain) on the step of imaging nucleic acid (such as imaging chain), wherein this imaging nucleic acid be attached to right Signal (that is, this non-cancellation of signal) is launched before connecing on nucleic acid.

In certain embodiments, method includes that a removal is attached to dock nucleic acid (such as provided herein Butt chain) on the step of imaging nucleic acid (such as imaging chain), wherein use the core not comprising quencher This imaging nucleic acid is removed in acid.

Stating in each of front in method, docking nucleic acid (including butt chain) can be that strand docks nucleic acid or right Chain link, or it can be double-strand docking nucleic acid or butt chain, or it can be partially double stranded docking core Acid or butt chain (such as, containing a strand and a double stranded region).

In certain embodiments, multiple target identification divisions (including multiple binding partners) are being used In the case of, the plurality of can contact with sample simultaneously and therefore contact with target interested.Target identification Partly (such as binding partners) need not sequentially contact with sample, although they are possible.

These distinct methods promotion rotating disk Laser Scanning Confocal Microscopes carry out high flux imaging.According to estimates, one The full cell 3D imaging process of secondary a kind of color will averagely need about 30 seconds.The method permission compatibility 10X or 20X object lens carry out imaging to large area (such as, up to mm scale).About 30-50 can be realized micro- The imaging depth of rice.Provided herein method have been used for dye actin, Ki-67, clathrin, Cytokeratin etc. (data are not shown).

Binding partners

These methods use the combination being coupled to nucleic acid (such as, docking nucleic acid (such as butt chain)) to join Even body.These can be referred to here as binding partners-nucleic acid conjugates (" BP-NA conjugate ").They May be additionally referred to as the target identification division of sequence mark.As used in this, " binding partners-nucleic acid is even Connection thing, " or " BP-NA conjugate, " refer to that a kind of connection is (such as, sub-by a kind of N-hydroxysuccinimidyl acyl Amine (NHS) joint) to the molecule of single-chain nucleic acid (such as, a DNA) butt chain.

The binding partners of conjugate can be for target interested (such as a, biomolecule (such as, protein or nucleic acid)) there is any part (such as, antibody or fit) of affinity. In certain embodiments, binding partners is a kind of protein.Comprise a kind of albumen being connected to butt chain The BP-NA-conjugate of matter (or peptide) can be referred to here as " protein-nucleic acid conjugate " or " protein-NA Conjugate." including but not limited to for the example of protein of conjugate of the present invention: antibody is (such as, single Clonal antibody), antigen binding antibody fragment (such as, Fab fragment), receptor, peptide and peptide fit. Other binding partners can use according to the present invention.Such as, imagine by electrostatic (such as, at this Electrostatic particle), hydrophobicity or magnetic (such as, magnetic-particle) interacts the knot being attached on target Close gametophyte.

As used in this, " antibody " includes full length antibody and its any Fab (example As, " antigen-binding portion thereof ") or strand.Term " antibody " includes but not limited to that one comprises and passes through disulfide bond Interconnective at least two weight (H) chain and the glycoprotein of two light (L) chains, or its antigen-binding portion Point.Antibody can be polyclone or monoclonal；Xenogenesis (xenogeneic), allogeneic Or isogenic (syngeneic) (allogeneic)；Or its modified forms (such as, humanization , chimeric).

As used in this, " antigen-binding portion thereof " of an antibody refers to the reservation specificity knot of antibody One or more fragments of the ability closed on antigen.The antigen combined function of antibody can be by full length antibody Fragment perform.The example being covered by the binding fragment in " antigen-binding portion thereof " of term antibody includes (i) Fab fragment, a kind of by V_H、V_L、C_LAnd C_H1The monovalent fragment of domain composition；(ii) F (ab') 2 Section, a kind of bivalent fragment comprising two the Fab fragments connected at hinge region by disulphide bridges；(iii) one Plant by V_HAnd C_H1The Fd fragment of domain composition；(iv) a kind of by the V of the single armed of antibody_HAnd V_LDomain The Fv fragment of composition, (v) is a kind of by V_HDomain composition dAb fragment (Ward (Ward) et al., Natural (Nature) 341:544 546,1989)；And (vi) complementarity-determining region separated , or (vii) two or more CDR separated of optionally being connected by synthetic linker (CDR) One combination.Although additionally, the two of Fv fragment domain V_HAnd V_LIt is by independent gene code , but the two domain can use recombination method to be engaged by synthetic linker, this synthetic linker energy It is enough made to make wherein V_HAnd V_LDistrict's pairing is to form simple protein chain (the referred to as scFv of monovalent molecule (scFv)；See, e.g. Byrd (Bird) et al., science (Science) 242:423 426, 1988；With Houston (Huston) et al., institute of NAS periodical (Proc.Natl.Acad.Sci. USA)85:5879-5883,1988).This kind of single-chain antibody is also covered by " the antigen-binding portion of term antibody Point " in the range of.Use routine techniques well known by persons skilled in the art, it is thus achieved that these antibody fragments, and The serviceability of described fragment is screened according to the mode identical with complete antibody.

As used in this, " receptor " refers to be attached to part (such as, such as, peptide or little molecule The organic or inorganic compound of (such as, low-molecular-weight (< 900 dalton))) on cell derived Molecule (such as, protein).

As used in this, " peptide is fit " refers to a kind of variable peptide having and inserting constant scaffold protein The molecule of sequence (see for example, Bei Ensi (Baines) IC et al., today drug discovery (Drug Discov.Today)11:334–341,2006)。

In certain embodiments, the molecule of BP-NA conjugate is that a kind of nucleic acid is (such as, such as, a kind of Aptamer).As used in this, " aptamer " refer to one can be formed can specificity knot Two grades closed on protein or other cell targets (see with tiny RNA or the DNA molecular of tertiary structure Such as, Ni (Ni) X et al., current medical chemistry (Curr Med Chem) 18 (27): 4,206 4214, 2011).Therefore, in certain embodiments, BP-NA conjugate can be a kind of fit-nucleic acid coupling Thing.

Some embodiments of the present invention use target identification division to identify and labelling target.Target identification part Point it is the reagent of target interested in specific recognition sample.The example of target identification division includes such as The binding partners of those listed herein.Target identification division includes antibody, antibody fragment and such as The antibody derivatives of single-chain antibody, single chain Fv constructs territory, Fab domain, nano antibody etc., peptide, suitable Body and oligonucleotide are (such as, in order to detection sense during such as fluorescence in situ hybridization or FISH The nucleic acid of interest).

The docking nucleic acid of such as butt chain

Certain embodiments of the present invention can relate to dock nucleic acid.Docking nucleic acid includes docking as described in this Chain.Docking nucleic acid is to be attached to have the line on the nucleic acid (such as, imaging nucleic acid) of complementary series Property nucleic acid.Docking nucleic acid can comprise or can be by DNA, RNA or have other phosphate-sugar backbone (such as 2 '-O-methyl RNA, 2 '-fluorine (2 '-fluoral) RNA, LNA, XNA) or comprise non-phosphate ester-sugar The nucleic-acid like structures of the main chain (such as, peptide nucleic acid(PNA) and morpholino) of part is constituted.Core base can include sky The core base so occurred, such as adenine, thymus pyrimidine, guanine, cytosine, inosine and they Derivant, and non-natural occur core base, such as isoC, isoG, dP and dZ.When not tying When closing on its complementary imaging nucleic acid, docking nucleic acid can be strand and not have stable secondary structure. Alternately, docking nucleic acid can include the secondary structure (Fig. 7, top) of such as hairpin loop.Docking nucleic acid It can be a part (Fig. 7, bottom) for multi-stranded complex.

As used in this, " butt chain " refers to a kind of complementary imaging chain that can be stably coupled to it On single-chain nucleic acid (such as, DNA).Stable combination can be butt chain (and contrary imaging Chain) the result of length, or it can be that (such as, salt is dense at its lower actual conditions that hybridization occurs Degree, temperature etc.) result.In certain embodiments, the length of butt chain is about 20 to 60, or more Multiple nucleotide.Butt chain can be attached to (have identical sequence and in the same manner labelling) one Or on multiple identical imaging chain.

The imaging nucleic acid of such as imaging chain

Certain embodiments of the present invention can relate to imaging nucleic acid.Imaging nucleic acid includes imaging as described in this Chain.Imaging nucleic acid be can (1) via sequence-specific complementarity with dock nucleic acid interaction, and (2) raise a signal emission part by covalently or non-covalently interacting or multiple signal emission part is copied The nucleic acid of shellfish.As described in this, imaging nucleic acid can be straight or branched.One imaging nucleic acid can Multiple signal is raised via a paradigmatic structure (Fig. 8, top) or dendritic structure (Fig. 8, bottom) Emitting portion copies.It is, for example possible to use such as receiving zamia (Nazemi) A. et al., Bioconjugation The chemistry of thing: synthesize, characterize with biomedical applications (Chemistry of Bioconjugates:Synthesis, Characterization, and Biomedical Applications), Online release: on February 13rd, 2014) The method of middle discussion and the reference wherein provided come chemosynthesis polymerization or dendritic structure.Alternative Ground, can be by as such as at De Kesi (Dirks) R. et al., and institute of NAS prints, 2004；1010(43):15275-78；And at crow nurse (Um) S.H. et al., natural research step (Nat. Protocols)2006；Shown in 1:995-1000 (described reference is combined in this each via quoting) DNA hybridization forms polymerization or dendritic structure.

Imaging nucleic acid can comprise or can by DNA, RNA or have other phosphate-sugar backbone (such as 2 '- O-methyl RNA, 2 '-fluorine (2 '-fluoral) RNA, LNA, XNA) or comprise non-phosphate ester-sugar portion The nucleic-acid like structures of the main chain (such as, peptide nucleic acid(PNA) and morpholino) divided is constituted.Core base can include natural Occur core base, such as adenine, thymus pyrimidine, guanine, cytosine, inosine and they Derivant, and the core base that non-natural occurs, such as isoC, isoG, dP and dZ.

In certain embodiments, the length of imaging nucleic acid is about 30 to about 60, or more nucleotide, Length including 30,35,40,45,50,55 or 60 nucleotide.In certain embodiments, imaging core A length of 30 to 40,30 to 50,40 to 50,40 to 60 or 50 to 60 nucleotide of acid.

When being not bonded on its complementary docking nucleic acid, imaging nucleic acid can be strand and not have stable Secondary structure.Alternately, imaging nucleic acid can include secondary structure (Fig. 9, the top of such as hairpin loop Portion).Imaging nucleic acid can be a part (Fig. 9, bottom) for multi-stranded complex.

In certain embodiments, imaging chain can be self-quenching, and this shows that unconjugated imaging nucleic acid can Carry the quencher moiety being very close to signal emission part (such as, fluorogen).In order to realize this Individual purpose, imaging nucleic acid can be designed as taking the structure (Fig. 5) or one and half of a molecular beacon sample The structure (Fig. 6) of duplex.

This self-quenching change may be used for simplifying background and/or avoiding washing step.Additionally or can Alternatively, it is conventionally used for FISH, RNA blotting and southern blotting technique in conjunction with containing with imaging buffer Additive (such as, electronegative polymer (such as asuro and heparin)) in method with Just non-specific binding is reduced.

" signal emission part " is one and launches detectable signal under certain conditions as used in this The part of (such as photon, radiation, positron, electromagnetic wave and nuclear magnetic resonant).

As used in this, " imaging chain " is that a kind of length is about 30 to about 60, or more nucleoside The single-chain nucleic acid (such as, DNA) of acid.The imaging chain of the present invention is complementary to butt chain and stably ties Close on butt chain.Stable combination shows, imaging chain and butt chain are in the duration measured or extremely Lack 30 minutes or at least 60 minutes or at least 2 hours or more Kubo is held and is bonded to each other.This combination can With yes or no reversible or irreversible.

In certain embodiments, if nucleic acid keeps being bonded to each other (or at least) 30,35,40,45, 50,55 or 60 minutes (min), then it is assumed that be attached to (the such as imaging of imaging nucleic acid docking nucleic acid stability Chain) on.In certain embodiments, if nucleic acid keeps being bonded to each other (or at least) 30 to 60min, 30 to 120min, 40 to 60min, 40 to 120min or 60 to 120min, then it is assumed that docking nucleic acid is steady Surely it is attached on imaging nucleic acid.This combination may or may not be reversible, or may or may not be Irreversible.

As used in this, " in conjunction with " refer between at least two molecule owing to such as electrostatic, hydrophobic Property, ion and/or interaction of hydrogen bond, a kind of association the most in physiological conditions.

If two nucleic acid or the base pairing each other of nucleic acid structure territory or combination are so that via Watson-Crick (Watson-Crick) interact a kind of double-strandednucleic acid of formation, then they are each other " complementary ".

In certain embodiments, the nucleic acid (such as docking nucleic acid and imaging nucleic acid) of the present invention is with " perfect mutual Mend " it is bonded to each other, " perfect complementary " refers to 100% complementary (such as, 5 ' ATTCGC 3 ' is perfect mutually Mend in 5 ' GCGAAT 3 ').

The imaging chain of the present invention can with a kind of detectable labelling (such as, a fluorescent labeling, and because of This is considered as " fluorescently-labeled ") it is marked.Such as, in certain embodiments, imaging chain can Comprise at least one (that is, one or more) fluorogen.The example of fluorogen used according to the invention Include but not limited to: ton derivant (such as, fluoflavin, rhodamine, Oregon be green, Yihong with And Texas is red), Cyanine derivative thing (such as, cyanine, indole carbon cyanines, oxa-carbon cyanines, thia carbon Cyanines and merocyanine), naphthalene derivatives (such as, red sulphonyl and Pu Luodan (prodan) derivant), fragrant Legumin derivant, oxadiazole derivative (such as, pyridine radicals azoles, nitro benzodiazole and benzo Diazole), pyrene derivatives (such as, waterfall blue), oxazine derivatives (such as, Nile red, Buddhist nun sieve Blue, cresol-purple and piperazine 170), acridine derivatives (such as, proflavine, acridine orange and acridine Yellow), arylmethine (arylmethine) derivant (such as, auramine, crystal violet and peafowl Green) and tetrapyrrole derivant (such as, porphin, phthalocyanine and bilirubin).

Can be with such as enumerating at this or those a kind of detectable label known in the art carrys out covalently labelling Imaging nucleic acid including imaging chain.In some cases, the imaging nucleic acid including imaging chain can include 2, 3,4 or more detectable labellings (such as fluorogen).

Orthogonal imaging nucleic acid including imaging chain can comprise different labellings (such as a, red fluorescence Group, blue-fluorescence group or green fluorescence group), or they can all comprise identical labelling (example As, red fluorescence group), even if their nucleotide sequence is different.

The target identification division of the such as sequence mark of binding partners and butt chain conjugate

In certain embodiments, the BP-NA conjugate (such as, protein-nucleic acid conjugate) of the present invention Can comprise one (such as, covalently or non-covalently) binding partners is connected to dock middle-of-chain connect Head.This transition joint can comprise biotin and/or Succ-PEG-DSPE.Such as, in some embodiments In, an antibody and a butt chain can each be biotinylated and (that is, connect at least one biotin to divide Son) and be bound to streptavidin molecule in the middle of by biotin and be connected to each other.Its He can use according to the present invention in transition joint.In certain embodiments, such as its Middle molecule is nucleic acid Those embodiments in, it may not be necessary to transition joint.Such as, the butt chain of BP-NA conjugate is permissible It is that (such as, 5 ' or 3 ' prolong for the extension of a nucleic acid molecules (such as, such as a, aptamer) Stretch).Similar method can be used for producing the target identification division of sequence mark as provided here.

Multiple BP-NA conjugates (such as, protein-nucleic acid conjugate) and imaging chain are provided at this.Many Individual can be a colony of same species or different plant species.Multiple BP-NA conjugates of same species can Including being fully incorporated to identical target (such as, biomolecule) (such as, identical epi-position or regions/junctions structure Territory) on conjugate.On the contrary, multiple BP-NA conjugates of different plant species can include conjugate or The subgroup of conjugate, the subgroup of each conjugate or conjugate is attached in the different epi-positions on identical target Or be attached on different target.Multiple imaging chains of same species can include having identical nucleotide sequence and The imaging chain of identical fluorescent labeling (such as, Cy2, Cy3 or Cy4).On the contrary, different plant species is many Individual imaging chain can include having different IPs nucleotide sequence (such as, DNA sequence) and different fluorescent labeling (such as, Cy2, Cy3 or Cy4), or there is different IPs nucleotide sequence and identical fluorescent labeling (example As, be all Cy2) imaging chain.The number of the different plant species in given multiple BP-NA conjugates Mesh is by the number of binding partners (such as, antibody) and has the butt chain of different IPs nucleotide sequence The restriction of the number of (and therefore complementary imaging chain).In certain embodiments, multiple BP-NA couplings Thing (such as, protein-nucleic acid conjugate) comprises at least 10,50,100,500,1000,2000, 3000、4000、5000、10⁴、50000、10⁵、10⁵、10⁶、10⁷、10⁸、10⁹、10¹⁰、10¹¹ Individual BP-NA conjugate.Similarly, in certain embodiments, multiple fluorescently-labeled imaging chains comprise to Few 10,50,100,500,1000,2000,3000,4000,5000,10⁴、50000、10⁵、 10⁵、10⁶、10⁷、10⁸、10⁹、10¹⁰、10¹¹Individual fluorescently-labeled imaging chain.In some embodiments In, multiple containing 1 to about 200 or the BP-NA conjugate of more different plant species and/or imaging chain. Such as, multiple can containing at least 1,2,3,4,5,6,7,8,9,10,15,20,25, 30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、125、 150,175,200 or more different species.In certain embodiments, multiple containing less than about 5 BP-NA conjugate and/or imaging chain to about 200 different plant species.Such as, multiple can containing is less than 5、6、7、8、9、10、15、20、25、30、35、40、45、50、55、60、65、70、 75,80,85,90,95,100,125,150,175 or 200 different species.These embodiments It is applicable to the target identification division of sequence mark as provided here.

Signal or the inactivation of imaging nucleic acid

In order to be implemented as nucleic acid inactivates in some of method provided herein, can be by such as but not limiting In a liter high-temperature；Reduce the concentration of counter ion (such as, free Mg++)；Introduce or increase denaturant The concentration of (such as Methanamide, carbamide, DMSO etc.)；And chemically, photochemically or enzymatic ground Crack, modify or be degraded into as chain, or the means of their combination in any, imaging nucleic acid (is included Imaging chain) from target identification division (including binding partners), remove (Fig. 3).

In order to be implemented as in some of method provided herein as nucleic acid inactivate, can by remove and/or Person modifies this signal emission part and is not removed from butt chain by whole nucleic acid moieties of imaging nucleic acid and inactivate These imaging nucleic acid (include imaging chain).(Fig. 4 .)

As an example, going of imaging nucleic acid can be promoted by imaging chain is cracked into some Remove.In certain embodiments, imaging nucleic acid comprises a chemical cleavable part, this chemistry cleavable portion Divide and can act on the compound in this cleavable part by introducing and crack.This type of chemistry cleavable The example of part includes but not limited to can be by the allyl group (week of some reagent based on Pd cracking (Ju) J. et al., National Academy of Sciences, on December 26th, 2006；103 (52): 19635-40, this ginseng Examine and be incorporated herein by reference)；The nitrine that can be cracked by some reagent based on phosphorus (such as TCEP) Base group, (Guo (Guo) J et al., National Academy of Sciences, on July 8th, 2008； 105 (27): 9145-50, this reference is incorporated herein by reference)；Can be by the bridge of reagent cracking based on silver Connect thiophosphate (Ma Ge (Mag) M. et al., nucleic acids research (Nucleic Acids Res), 1991 On April 11, in；19 (7): 1437-41, this reference is incorporated herein by reference)；Can by such as DTT and The disulfide bond of the reducing agent cracking of TCEP；And can be by the various nucleopilic reagents of such as hydroxide and imidazoles The ribose of cracking.

In certain embodiments, this imaging nucleic acid comprise one can be (such as, sudden and violent by UV photochemically Dew) crack can photodestruciton joint.In certain embodiments, this imaging nucleic acid contain one can be by one Plant the part of enzymatic lysis.The example of this type of enzymatic cleavable part includes but not limited to can be by various ribose The ribonucleotide of nuclease cleavage；Can be by such as USER (New England biology laboratory (New England Biolabs)) enzyme combination cracking BrdU；And can be by sequence-specific nickase Or the restriction enzyme site of restriction enzyme enzymatic lysis.In certain embodiments, this restricted enzyme Can be cleaved into as nucleic acid and docking nucleic acid.In still other embodiments, can be by by imaging nucleic acid It is modified into and a kind of makes docking nucleic acid combine to form (or the dissolving reduced of a stability with reduction Temperature) the form of duplex promote the removal of imaging nucleic acid.

As an example, this imaging nucleic acid comprises can be with the diphenyl diimide of photoisomerization, wherein different isomerization Body differently affect imaging nucleic acid for docking nucleic acid bond strength (shallow natural pond (Asanuma) H. et al., Applied chemistry English world version (Angew Chem Int Ed Engl), July 16 calendar year 2001； 40 (14): 2671-2673, this reference is incorporated herein by reference).In certain embodiments, this imaging core Acid comprises a kind of BrdU, and wherein uracil group can be cracked by uracil-DNA glycosylase.Going After uracil, the bond strength of imaging chain weakens.

Alternately, can by by between imaging nucleic acid and signal emission part joint (if there is Such joint) crack the removal realizing signal emission part.Chemical substance described here can also For this purpose.

This signal emission part can be realized by chemically or photochemically modifying signal emission part Inactivation.Such as, when this signal emission part is a fluorogen, can by chemical reagent (such as Such as hydrogen peroxide, Ge Disi (Gerdes) M. et al., National Academy of Sciences on July 16th, 2013； 110 (29): 11982-87, this reference is incorporated herein by reference) bleached or it is carried out photobleaching (such as, use as at Shu Baite (Schubert) W. et al., Nature Biotechnol (Nat. Biotech.)2006；Soft multi-wavelength excitation described in 24:1270-78, this reference is hereby incorporated by This).

As in the art it will be appreciated that, " photobleaching " refers to a dyestuff or the photochemistry of fluorophore molecule Change, so that it can not send fluorescence.This is by the covalency bond cleavage between fluorogen and surrounding molecular Solve or nonspecific reaction and cause.In certain embodiments, the intensity that can be exposed by reduction or Time interval, by increasing the concentration of fluorogen, by reducing the frequency of input light and therefore reducing its light Sub-energy, or more firm fluorogen (the such as Alexa Fluors of bleaching it is less susceptible to by employing Or DyLight Fluors), control the loss of activity caused by photobleaching.See, e.g., Ghauharali R. et al., microscopy magazine (Journal of Microscopy) 2001；198:88- 100；With Green (Eggeling) C. et al., analytical chemistry (Analytical Chemistry) 1998；70:2651-59.

Therefore, photobleaching can be used for removing, modify or eliminating in some cases from signal emission part Signal.Can be by fluorogen being exposed to the wavelength of the light with applicable wavelength, energy and persistent period Carry out photobleaching, in order to forever and irreversibly eliminate fluorogen and launch the ability of signal further.Light Bleaching technology is as known in the art.

Also find, and usually less than under the wide range temperature (that is, 0 DEG C to 37 DEG C) of physiological temp It is attached on their target and slightly changing (that is, from 10mM's to 1M of salinity can be stood Monovalent cation concentration；From the divalent cation concentration of 0 to 10mM) antibody different, short nucleic acid hybridization Affinity depend on temperature and salinity.Such as, at 23 DEG C, there is 500mM [Na+] and 10 Under mM [Mg++] concentration, ssDNA 5 '-CATCTAAAGCC-3 ' and its reverse complemental chain 5 '- Prediction dissociation constant between GGCTTTAGATG-3 ' (uses the ginseng with reference to PMID 15139820 general introduction Manifold) be～90pM.In other words, with this understanding, this combination is the strongest.At 37 DEG C, Under having 150mM [Na+] and 0mM [Mg++], this prediction dissociation constant of ssDNA pair is up to ～500nM.In other words, with this understanding, this combination is the most weak.Often dissociating of both conditions Number changes nearly 4 orders of magnitude, while it is contemplated that most of antibody is the most strongly attached to it Target on.Similar trend (Figure 10) is observed for other DNA sequence.As one additionally Example, image-forming condition can be 23 DEG C, have 500mM [Na+] and 10mM [Mg++], and contaminate Material inactivation condition can be 37 DEG C, have 150mM [Na+] and 0mM [Mg++].

In certain embodiments, analyzed sample is cell, the tissue slice or raw from living cultivated Other samples of object.

In certain embodiments, this sample is that fixing (including the most fixing) is a surface of solids (example As, glass slide or coverslip) on dissociated cell.Such as, sample can be the cell in blood. Such as, sample can containing circulate in blood cancerous cell (also referred to as circulating tumor cell, or CTC).Sample can be the cell of growth in suspension.Sample can be to dissipate from a solid tissue The cell broadcast.

Sample

" sample " can include cell (or a cell), tissue or such as blood (serum and/or blood Slurry), urine, seminal fluid, lymph fluid, cerebrospinal fluid or the body fluid of amniotic fluid.Sample be available from (or source In) any source, include but not limited to the mankind, animal, antibacterial, virus, microorganism and plant. In certain embodiments, sample is cell lysate or Tissue Lysates.Sample also can be containing from one The mixture of the material of source or separate sources.Sample can be an area of space or volume (such as, Hole in grid on one array or a plate or dish).In certain embodiments, sample includes one Individual or multiple target, one or more BP-NA conjugate and one or more imaging chain.These are thin Born of the same parents can be (or dissociating) cell disseminated.

Target

" target " is desirable to observe or quantify and exist any part of its binding partners.Real at some Executing in example, target can be naturally occurring with right and wrong.In certain embodiments, target can be a kind of biological Molecule.As used in this, " biomolecule " is any molecule that can be produced by a live organism, Including big macromole, such as protein, polysaccharide, lipid and nucleic acid (such as, DNA and RNA (such as mRNA)), and little molecule, such as primary metabolite, secondary metabolite and Natural product.The example of biomolecule includes but not limited to: DNA, RNA, cDNA or stand inverse The DNA product of the RNA transcribed, A23187 (calcimycin, Calcium ionophore), avilamycin, pine Fragrant acid, acetic acid, acetylcholine, actin, actinomycin D, adenosine, adenosine diphosphate (ADP), adenylic acid (AMP), adenosine triphosphate (ATP), adenyl cyclase, side Adonistal, epinephrine (Adrenaline), epinephrine (epinephrine), rush adrenal gland's skin Matter hormone (ACTH), aequorin (Aequorin), aflatoxin, agar, the third first bacterium Element, alanine, albumin, aldosterone, aleurone (Aleurone), α-poisonous fungus cyclic peptide, allantoin, third Alkene chrysanthemum ester, α-amanitin (α-Amanatin), aminoacid, amylase, anabolic steroid (Anabolic steroid), anethole, proangiotensin, anisomycin, vassopressin (ADH), arabinose, arginine, ascosin, ascorbic acid (vitamin C), Radix Asparagi acyl Amine, aspartic acid, ADMA, atrial natriuretic peptide (ANP), auxin, antibiosis Thing fibroin, nimbin A C35H44O16, bacteriocin, beauvericin, bicuculline, gallbladder are red Element, biopolymer, biotin (biotin), brefeldin A, brassinolide, horse Semen Strychni alkali, cadaverine, caffeine, calciferol (vitamin D), calcitonin, calmodulin, CaM, calcium adjust egg In vain, calprotectin, Camphora-(C10H16O), cannabinol, capsaicin, carbohydrase, carbon hydrate Thing, carnitine, carrageenin, casein, caspase, cellulase, cellulose- (C6H10O5), cerulenin, cetrimonium bromide (Cetrimide)-C19H42BrN, Herba Chelidonii are red Alkali, chromomycin A3, chaperone, chitin, Chloralosane, chlorophyll, cholecystokinin (CCK), cholesterol, choline, chondroitin sulfate, cinnamic aldehyde, citral, citric acid, Fructus Citri tangerinae are mould Element, citronellal, citronellol, citrulline, cobalamine (vitamin B12), coenzyme, ubiquinone, autumn waters--limid eyes Celestial alkali, collagen, coniine, corticosteroid, corticosterone, corticotropin releasing hormone (CRH), hydrocortisone, creatine, creatine kinase, crystallin, alpha-cyclodextrin, cyclodextrin glycosyl Transferring enzyme, cyclopamine, cyclopiazonic acid, cysteine, cystine, cytidine, cytochalasin, thin Born of the same parents relaxin E, cytochrome, cytochrome C, cytochrome c oxidase, cytochrome c peroxide Enzyme, cytokine, cytosine C4H5N3O, deoxycholic acid, DON (deoxynivalenol bacterium alkene Alcohol), desoxyribofuranose, deoxyribose, DNA (deoxyribonucleic acid) (DNA), dextran, paste Essence, DNA, dopamine, enzyme, ephedrine, epinephrine C9H13NO3, erucic acid CH3 (CH2) 7CH=CH (CH2) 11COOH, erythritol, erythropoietin (EPO), female Glycol, acetaminol, fatty acid, fibrin, fibronectin, folic acid (vitamin(e) M), rush ovum Bubble hormone (FSH), formaldehyde, formic acid, Formnoci, fructose, fumonisins B1, third kind of ball egg In vain, galactose, gamma globulin, γ-aminobutyric acid, gamma-butyrolacton, gamma-hydroxybutyric acid ester (GHB), Gastrin, gelatin, geraniol, globulin, glucagon, glucamine, glucose C6H12O6, glucose oxidase, glutelin, glutamic acid, glutamine, glutathion, glutelin, Glycerol (glycerol), glycine, glycogen, glycolic, glycoprotein, gonadotropin releasing hormone (GnRH), granzyme, GFP egfp, growth hormone, growth hormone releasing hormone (GHRH), GTP enzyme, guanine, guanosine, guanosine triphosphate (GTP) (+GTP), haptoglobin, Soviet Union Another name for, haemachrome, hemerythrin, hemocyanin, hemoglobin, hemoprotein, sulphuric acid second Acyl heparin, high density lipoprotein, HDL, histamine, histidine, histone, histone methyl shift Enzyme, HLA antigen, homocysteine, hormone, chorionic-gonadotropin hormone (hCG), human growth Hormone, hyaluronate, hyaluronidase, hydrogen peroxide, 5-hydroxymethyl cytosine, hydroxyl dried meat ammonia Acid, 5-hydroxy tryptamine, bipseudoindoxyl dye, indole, inosine, inositol, insulin, insulin-like growth factor Son, integral protein, intergrase, integral protein, intein, interferon, inulin, ionomycin, Ionone, isoleucine, iron-sulfur cluster, K252a, K252b, KT5720, KT5823, keratin, Kinases, Lactose enzyme, lactic acid, lactose, lanoline, lauric acid, leptin, leptomycin B, leucine, Lignin, limonene, linalool, linoleic acid, linolenic acid, lipase, lipid, lipid-anchored egg In vain, thioctamide, lipoprotein, low density lipoprotein, LDL LDL, interstitialcellstimulating hormone (ICSH) (LH), tomato red Element, lysine, lysozyme, malic acid, maltose, melatonin, memebrane protein, metalloprotein, gold Belong to sulfoprotein, methionine .beta.-[N-(3-hydroxy-4-pyridone), mithramycin A, ametycin, monomer, mycophenolic acid, Myoglobin, myosin, natural phenolic, nucleic acid, ochratoxin A, estrogen, oligopeptide, few mould Element, orcin, aricine element, ornithine, oxalic acid, oxidase, oxytocin, p53, PABA, purple China fir alcohol, Palmic acid, pantothenic acid (vitamin B5), parathyroid hormone (PTH), paraprotein, Moses Ichthyotoxin peptide, parthenolide, patulin, gill fungus penicillin .gamma.-keto-.beta.-methoxy-.delta.-methylene-.DELTA..alpha.-hexenoic acid., penicillin, penicillium sp tremble Element A, peptidase, pepsin, peptide, Aminomycin, peripheral membrane protein, Perosamine, phenethylamine, benzene Alanine, phosphagen, phosphatase, phospholipid, phenylalanine, phytic acid, phytohormone, polypeptide, many Phenol, polysaccharide, porphyrin, Protein virus, Progesterone, prolactin antagonist (PRL), proline, propanoic acid, milt (dimension is raw for albumen, protease, protein, albuminoid, putrescine, pyrethrin, pyridoxol or pyridoxamine Element B6), pyrrolysine, acetone acid, quinone, radicicol, Raffinose, feritin, retinal, Retinol (vitamin A), rhodopsin (rhodopsin), riboflavin (vitamin B2), furan core Sugar, ribose, ribozyme, ricin, RNA-ribonucleic acid, RuBisCO, safrole, bigcatkin willow Aldehyde, salicylic acid, red phenol-A (Salvinorin-A) C23H28O8, saponin, secretin, seleno half Cystine, selenomethionine, selenoprotein, serine, serine kinase, serotonin, excrement are smelly Element, signal recognition particle, somatostatin, sorbic acid, Squalene, D-82041 DEISENHOFEN, stearic acid, miscellaneous Color aspergillin, sterol, brucine (Strychnine), sucrose (sugared), saccharide (typically come Say), superoxides, T2 toxin, tannic acid, tannic acid, tartaric acid, taurine, Fugu ocellatus toxin, strange Monellin, topoisomerase, tyrosine kinase, taurine, testosterone, tetrahydrocannabinol (THC), Fugu ocellatus toxin, thapsigargin, thaumatin, thiamine (vitamin B1) C12H17ClN4OS HCl, threonine, thrombopoietin, thymidine, thymus pyrimidine, three nitrogen bacterium Element C, thyrotropin (TSH), thyrotrophin-releasing hormone (TRH), thyroxine (T4), tocopherol (vitamin E), topoisomerase, 3 (T3), cross-film Receptor, Trichostatin A, trophic hormone, trypsin, tryptophan, tubulin, tunicamycin, cheese ammonia Acid, ubiquitin, uracil, carbamide, urase, uric acid C5H4N4O3, uridnine, valine, figured silk fabrics Amino doxycycline, vanadium binding albumen (Vanabins), vassopressin, tremorgenic mycotoxin (Verruculogen), vitamin (in general), vitamin A (retinol), vitamin B, Vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (nicotinic acid or nicotinic acid), Adenine phosphate (adenine), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxol or pyridoxamine), Vitamin B12 (cobalamine), vitamin C (ascorbic acid), vitamin D (calciferol), dimension are raw Element E (tocopherol), vitaminF, biotin (biotin), vitamin K (naphthoquinone), vitamin M (folic acid), wortmannin and xylose.

In certain embodiments, a target can be a protein target, the most such as, Yi Zhongxi The protein (such as, intracellular or memebrane protein) of born of the same parents' environment.The example of protein includes but does not limits In: fibrous proteins, such as cyto skeletal protein (such as, actin, arp2/3, hat egg In vain, dystrophin, FtsZ, keratin, myosin, nebulin, spectrin, tau egg In vain, titin, tropomyosin, tubulin and collagen), and extracellular matrix protein (such as, collagen, elastin laminin, f spondin (f-spondin), pick up mound element (pikachurin) and fibronectin)；Globular protein, such as plasma proteins (such as, blood Clear Amyloid P component and serum albumin), (such as, complement protein, C1-press down thrombin Preparation and C3-invertase, Factor IX, FXIII, fibrin, protein C, protein S, Protein Z, protein Z associated protein enzyme inhibitor, thrombin, von Willebrand factor), with And acute phase protein, such as CRP；Hemoprotein；Cell adhesion protein (such as, calcium Mucin, ependyma albumen (ependymin), integral protein, Ncam and selection albumen)；Across Film transport protein matter (such as, CFTR, glycophorin D and mix enzyme (scramblase)), such as (such as, ligand-gated ion channel, such as nAChR and GABAa are subject to ion channel Body, and voltage gated ion channel, such as potassium, calcium and sodium channel), transport in the same direction/reversely transport egg White matter (such as, glucose transporter)；Hormone and somatomedin (such as, epidermal growth factor (EGF), fibroblast growth factor (FGF), VEGF (VEGF), peptide Parahormone (such as insulin, insulin like growth factor and oxytocin) and steroid hormone are (all Such as androgen, estrogen and Progesterone)；Receptor, such as (such as, G-protein coupling is subject to transmembrane receptor Body, rhodopsin) and intracellular receptor (such as, estrogen receptor)；DNA binding protein dna (example As, histone, protamine, CI albumen)；Transcriptional (such as, c-myc, FOXP2, FOXP3, MyoD and P53)；Immune system protein matter (such as, immunoglobulin, Main Tissues Compatibility antigen and φt cell receptor)；Nutrient storage/transport protein matter (such as, ferritin)； Chaperone；And enzyme.

In certain embodiments, target can be a kind of nucleic acid target, such as, such as, cellular environment Nucleic acid.As used herein about target, butt chain and imaging chain, " nucleic acid " refers to have any The polymer form of the nucleotide of length, such as deoxyribonucleotide or ribonucleotide, or they Analog.Such as, nucleic acid can be the DNA product of DNA, RNA or the RNA standing reverse transcription. The limiting examples of nucleic acid includes: gene or the coding of genetic fragment or noncoding region, by linkage analysis The locus of restriction, exon, intron, messenger RNA (mRNA), transfer RNA, ribosome RNA, ribozyme, cDNA, recombinant nucleic acid, branched nucleic acids, plasmid, carrier, have any sequence point From DNA, separation RNA, nucleic probe and the primer with any sequence.Other examples of nucleic acid Include but not limited to: cDNA, fit, peptide nucleic acid(PNA) (" PNA "), (one has shortening to 2'-5'DNA The synthetic material of main chain, this main chain has the base intervals of the A conformation of coupling DNA；2'-5'DNA is usual Will not with the DNA hybridization for B form, but it will easily with RNA hybridize), lock nucleic acid (" LNA ") and have modify main chain nucleic acid (such as, the base modification of naturally occurring nucleic acid Or sugar-modified form).Nucleic acid can comprise the nucleotide of modification, the most methylated nucleotide and nucleotide Analog (purine be in the art with " similar " form of pyrimidine known to).If there is to nucleoside The modification of acid structure, then carried out before or after assembling polymer.Nucleic acid can be a kind of strand, Double-strand, part strand or partially double stranded DNA or RNA.

In certain embodiments, nucleic acid (such as, nucleic acid target) is naturally-occurring.As made at this , " naturally occurring " refers to a kind of in the case of not having human intervention, is present in and deposits in nature Organism or virus in nucleic acid.In certain embodiments, nucleic acid is naturally occurring in a kind of organism Or in virus.In certain embodiments, nucleic acid be genomic DNA, messenger RNA, ribosomal RNA, Microrna, front Microrna (pre-micro-RNA), former Microrna (pro-micro- RNA), viral DNA, viral RNA or piwi-RNA.In certain embodiments, nucleic acid target is not A kind of synthetic DNA nanostructured (such as, comprising is interacted by Watson-Crick hybridize each other with Form the two dimension (2-D) of two or more nucleic acid of 2-D or 3-D nanostructured or three-dimensional (3-D) DNA nanostructure).

Nucleic acid butt chain described here and imaging chain can be nucleic acid described above (such as, DNA, RNA, modification of nucleic acids, nucleic acid analog, naturally occurring nucleic acid, nucleic acid) in any one Kind.

Compositions

Compositions comprises at least one or the BP-of at least two (such as, the multiple) present invention provided herein NA conjugate (such as, protein-nucleic acid conjugate).BP-NA conjugate can be coupled to one interested The fluorescently-labeled one-tenth that target (such as, biomolecule) is upper and/or stable bond to is complementary As on chain.Compositions can comprise the BP-NA conjugate of multiple same species or different plant species.Real at some Execute in example, a kind of compositions can comprise at least 10,50,100,500,1000,2000,3000, 4000、5000、10⁴、50000、10⁵、10⁵、10⁶、10⁷、10⁸、10⁹、10¹⁰、10¹¹Individual BP- NA conjugate.In certain embodiments, a kind of compositions can comprise at least 10,50,100,500, 1000、2000、3000、4000、5000、10⁴、50000、10⁵、10⁵、10⁶、10⁷、10⁸、 10⁹、10¹⁰、10¹¹The fluorescently-labeled imaging chain of individual complementation.In certain embodiments, a kind of compositions can Containing 1 to about 200 or the BP-NA conjugate of more different plant species and/or imaging chain.Such as, a kind of Compositions can containing at least 1,2,3,4,5,6,7,8,9,10,15,20,25,30, 35、40、45、50、55、60、65、70、75、80、85、90、95、100、125、150、 175,200 or more different species.In certain embodiments, a kind of compositions can be containing less than about 5 BP-NA conjugate and/or imaging chain to about 200 different plant species.Such as, a kind of compositions can contain Less than 5,6,7,8,9,10,15,20,25,30,35,40,45,50,55,60,65, 70,75,80,85,90,95,100,125,150,175 or 200 different species.

The number that should be appreciated that the complementary fluorescently-labeled imaging chain in compositions is smaller than, equal to or More than the number of BP-NA conjugate in compositions.

Test kit

The present invention further provides the test kit including one or more instant component.Test kit can wrap Include, such as, BP-NA conjugate and/or fluorescently-labeled imaging chain.Test kit may also include for producing BP-NA conjugate or for being marked as the component as chain.Such as, test kit can include binding partners (such as, antibody), butt chain and transition joint (such as, such as, biotin and streptavidin Protein molecular) and/or imaging chain.Test kit may be used for the mesh that those skilled in the art substantially know , including those described above.

Test kit can include other reagent, and such as, is used for carrying out the buffer of hybridization.Reagent Box may also include for use reagent constituents and/or for make and/or use BP-NA conjugate and/ Or the description of labeled imaging chain.

Application

The BP-NA conjugate (such as, protein-nucleic acid conjugate or antibody-nucleic acid conjugate) of the present invention Especially in which can use in any mensuration of existing target detection technology and use.

Typically, mensuration includes following detection assay: include diagnostic assay, prognosis mensuration, patient-monitoring Mensuration, Screening test, biological warfare mensuration, forensic analysis mensuration, prenatal gene diagnosis mensuration etc..This survey Surely can be a kind of external test or a kind of in vivoassay.The present invention provides the advantage that and can use this The method of invention analyzes many different targets from a simple sample simultaneously, even ought wherein this type of target Mark uses prior art formation method spatially can not differentiate (and the most indecipherable) Time.This permission such as carries out several diagnostic test on a sample.

BP-NA conjugate can be also used for observing a region or district simply.

Can be applied to the method for the present invention analyze the sample deriving from or deriving from patient, in order to determine one Whether the cell type of pathological changes is present in this sample and/or carries out this disease by stages.Such as, may be used To measure a blood sample according to any method described here, in order to determine a canceration in sample The existence of the label of cell type and/or amount, thus diagnose this cancer or by stages.

Alternately, these methods described here may be used for by determining antibacterial or disease in sample respectively Existence and/or the amount of the label of poison diagnose pathogenic infection, such as, Intracellular bacterial and virus draw The infection risen.Therefore, the target using the compositions and methods of the invention detection can be patient indicia's thing (such as a kind of cancer markers) or the label infected by a kind of outside agent, such as antibacterial or virus Label.

The quantitative imaging method of the present invention can be used for such as, quantifies its abundance instruction physiological status or disease is sick The target (such as, target biomolecule) of shape (such as, is upward or downward as a kind of morbid state The blood marker of result).

It addition, the compositions and methods of the invention can be used for providing the prognosis letter aiding in determining whether patient procedure Breath.For example, it is possible to quantify for tumor exactly from even one the little sample from patient The amount of individual concrete label.For some disease (as breast carcinoma), some protein (such as Her2- Neu) overexpression shows to need a kind of more positive course for the treatment of.

The method of the present invention can be also used for determining that disturbance (includes compound, sudden change, temperature change, life The change of long hormone, somatomedin, disease or condition of culture) for the impact of different targets, thus Identify that it exists, lacks or the target of the level concrete biological condition of instruction.In certain embodiments, originally Invention is for illustrating and find component and the path of morbid state.Such as, be present in a diseased tissue with The comparison of the amount of the target in " normally " tissue allows explanation to relate to the important target of disease, thus identifies use In finding/screen may be used for the target of the Novel drug candidates for the treatment of disease.

The sample analyzed can be a biological sample, such as blood, saliva, lymph, mucus, excrement Just, urine etc..This sample can be a kind of environmental sample, such as water sample, air sample, food sample Product etc..One or more components fixed that can use association reaction carry out this mensuration.Therefore, target Mark or BP-NA conjugate can be fixing.Can use association reaction revocable one or more Component carries out this mensuration.In view of being provided by BP-NA conjugate and the fluorescently-labeled imaging chain of the present invention Multiplexing potentiality, these mensuration can relate to substantially simultaneously detect the multiple targets in a sample. As an example, a kind of mensuration can be used for a kind of concrete cell type of detection (such as, based on a kind of special Fixed cell surface receptor) and a kind of concrete gene mutation in this concrete cell type.With this side Formula, as an example, end user may be able to determine that how many cells of a kind of particular type carry sense The sudden change of interest.

Different embodiments

Present disclosure provides and includes but not limited to multiple embodiments of following numbering embodiment:

1. a method, including

(1) make a sample needing to carry out testing for the existence of one or more targets with one or more Target specificity binding partners contact, the most each target specificity binding partners be connected to one right Chain link, and wherein there is the most homospecific target specificity binding partners be connected to different docking core Chain,

(2) unconjugated target specificity binding partners is optionally removed,

(3) the labeled imaging making this sample and have the nucleotide sequence being complementary to a butt chain links Touch,

(4) unconjugated labeled imaging chain is optionally removed,

(5) by this sample imaging to detect position and the number of the labeled imaging chain combined,

(6) quencher is from the signal of the labeled imaging chain combined, and

(7) repeat step (3)-(6), use one every time for all other labeled imaging chains There is the labeled imaging chain of unique nucleotide sequence.

2. as described in Example 1 method, wherein this sample in step (1) with more than one target Mark specific binding partner contact.

3. the method as described in embodiment 1 or 2, wherein this target specificity binding partners is a kind of anti- Body or antibody fragment.

4. the method as according to any one of embodiment 1-3, wherein these labeled imaging chains are by identical Ground labelling.

5. the method as according to any one of embodiment 1-3, wherein these labeled imaging chains are each self-contained One different labelling.

6. the method as according to any one of embodiment 1-5, wherein these labeled imaging chains are fluorescence marks The imaging chain of note.

7. the method as according to any one of embodiment 1-6, wherein these one or more targets are albumen Matter.

8. the method as according to any one of embodiment 1-7, wherein this sample is cell, cell lysate Or Tissue Lysates.

9. the method as according to any one of embodiment 1-8, wherein uses copolymerization Jiao or epi-fluorescence micro- Microscopy in step (5) by this sample imaging.

10. the method as according to any one of embodiment 1-9, wherein the quencher signal in step (6) includes Photobleaching.

11. 1 kinds of compositionss, comprise:

The sample being attached on more than one target specificity binding partners, each combination spouse Body is attached on a butt chain, and

At least one is stably coupled to the butt chain on a labeled imaging chain.

12. 1 kinds of methods, including

(1) make a sample needing to carry out testing for the existence of one or more targets with one or more Target specificity binding partners contact, the most each target specificity binding partners be connected to one right Connect nucleic acid, and wherein there is the most homospecific target specificity binding partners be connected to different docking Nucleic acid,

(2) unconjugated target specificity binding partners is optionally removed,

(3) make this sample and there is the labeled imaging nucleic acid being complementary to a nucleotide sequence docking nucleic acid Contact,

(4) unconjugated labeled imaging nucleic acid is optionally removed,

(5) by this sample imaging to detect position and the number of the labeled imaging nucleic acid combined,

(6) combining labeled imaging nucleic acid is removed from docking nucleic acid, and

(7) repeat step (3)-(6), use one to come relative to all other labeled imaging nucleic acid every time Say the labeled imaging nucleic acid with unique nucleotide sequence.

13. methods as described in Example 12, wherein this sample in step (1) with more than one target Mark specific binding partner contact.

14. methods as described in embodiment 12 or 13, wherein this target specificity binding partners is a kind of Antibody or antibody fragment.

15. methods as described in embodiment 12 or 13, wherein this target specificity binding partners is natural Or the part of through engineering approaches, little molecule, fit, peptide or oligonucleotide.

16. methods as according to any one of embodiment 12-15, wherein these labeled imaging nucleic acid by Labelling in the same manner.

17. methods as according to any one of embodiment 12-15, wherein these labeled imaging nucleic acid are each Comprise a different labelling.

18. methods as according to any one of embodiment 12-17, wherein these labeled imaging nucleic acid are glimmering The imaging nucleic acid of signal.

19. methods as according to any one of embodiment 12-18, wherein these one or more targets are albumen Matter.

20. methods as according to any one of embodiment 12-19, wherein this sample is cell, cell cracking Thing or Tissue Lysates.

21. methods as according to any one of embodiment 12-20, wherein use copolymerization burnt or epi-fluorescence show Micro-microscopy in step (5) by this sample imaging.

22. methods as according to any one of embodiment 12-21, wherein these labeled imaging nucleic acid are logical Cross and reduce salinity, interpolation denaturant or improve what temperature was removed from docking nucleic acid.

23. methods as described in Example 22, wherein this salt is Mg++.

24. methods as described in Example 22, wherein this denaturant is Methanamide, carbamide or DMSO.

25. methods as according to any one of embodiment 12-21, wherein in the existence of a competition nucleic acid Under, by strand displacement, labeled imaging nucleic acid is not removed from docking nucleic acid.

26. methods as according to any one of embodiment 12-21, wherein by chemical method, photochemical method or Enzymatic method cracking, labeled imaging nucleic acid of modifying or degrade, go labeled imaging nucleic acid from docking nucleic acid Remove.

27. methods as according to any one of embodiment 12-21, are wherein attached to when labeled imaging nucleic acid Time on the docking nucleic acid of its correspondence, there is not strand district in imaging nucleic acid or docking nucleic acid.

28. methods as according to any one of embodiment 12-21, wherein this labeled imaging nucleic acid is not certainly Cancellation.

29. methods as according to any one of embodiment 12-28, wherein this unconjugated docking nucleic acid is portion Divide double-strand.

30. methods as according to any one of embodiment 12-28, wherein this unconjugated imaging nucleic acid is portion Divide double-strand.

31. methods as according to any one of embodiment 12-28, wherein this imaging nucleic acid is a molecule letter Mark or include a hairpin secondary structures.

32. methods as according to any one of embodiment 12-27 and 29-31, wherein this imaging nucleic acid is one Individual molecular beacon or include the hairpin secondary structures of a self-quenching.

33. methods as according to any one of embodiment 12-28, wherein this imaging nucleic acid is one and half double-strands Body.

34. methods as described in embodiment 33, wherein this half duplex is self-quenching.

35. methods as according to any one of embodiment 12-34, wherein this docking nucleic acid includes a hair clip Secondary structure.

36. methods as according to any one of embodiment 12-35, wherein this imaging nucleic acid is by a tree Dendritic morphology or a paradigmatic structure are attached in multiple signal emission part.

34. 1 kinds of methods, including

(2) unconjugated target specificity binding partners is optionally removed,

(4) unconjugated labeled imaging nucleic acid is optionally removed,

(6) not exclusively remove imaging nucleic acid inactivate knot by removing or modify their signal emission part The labeled imaging nucleic acid closed, and

35. methods as described in embodiment 34, wherein this sample in step (1) with more than one target Mark specific binding partner contact.

36. methods as described in embodiment 34 or 35, wherein this target specificity binding partners is a kind of Antibody or antibody fragment.

37. methods as described in embodiment 34 or 35, wherein this target specificity binding partners is natural Or the part of through engineering approaches, little molecule, fit, peptide or oligonucleotide.

38. methods as according to any one of embodiment 34-37, wherein these labeled imaging nucleic acid by Labelling in the same manner.

39. methods as according to any one of embodiment 34-37, wherein these labeled imaging nucleic acid are each Comprise a different labelling.

40. methods as according to any one of embodiment 34-39, wherein these labeled imaging nucleic acid are glimmering The imaging nucleic acid of signal.

41. methods as according to any one of embodiment 34-40, wherein these one or more targets are albumen Matter.

42. methods as according to any one of claim 34-41, wherein this sample is that cell, cell split Solve thing or Tissue Lysates.

43. methods as according to any one of embodiment 34-42, wherein use copolymerization burnt or epi-fluorescence show Micro-microscopy in step (5) by this sample imaging.

44. methods as according to any one of embodiment 34-43, wherein this unconjugated docking nucleic acid is portion Divide double-strand.

45. methods as according to any one of embodiment 34-43, wherein this unconjugated imaging nucleic acid is portion Divide double-strand.

46. methods as according to any one of embodiment 34-45, wherein this imaging nucleic acid is a molecule letter Mark or include a hairpin secondary structures.

47. methods as according to any one of embodiment 34-45, wherein this imaging nucleic acid is a molecule letter Mark or include the hairpin secondary structures of a self-quenching.

48. methods as according to any one of embodiment 34-45, wherein this imaging nucleic acid is one and half double-strands Body.

49. methods as described in embodiment 48, wherein this half duplex is self-quenching.

50. methods as according to any one of embodiment 34-49, wherein this docking nucleic acid includes a hair clip Secondary structure.

51. methods as according to any one of embodiment 34-50, wherein this imaging nucleic acid is by a tree Dendritic morphology or a paradigmatic structure are attached in multiple signal emission part.

Equivalents

Although being described herein and shown several inventive embodiments, but those of ordinary skill in the art having held Easily the imagination for perform function and/or obtain in result and/or advantage described here one or more many Each in kind of other devices and/or structure, and this type of modification and/or amendment is considered to be is retouching at this In the range of the inventive embodiments stated.In more general terms, it will be appreciated by those of ordinary skill in the art that This all parameters, size, material and configuration described means as exemplary, and actual ginseng Number, size, material and/or configuration will depend upon which that invention is taught one or more used for content and specifically should With.Those skilled in the art simply uses normal experiment it will be recognized that or be able to confirm that tool described here Many equivalents of body inventive embodiments.It is therefore to be understood that foregoing embodiments is only by means of example Presenting, and in the range of appended claims and its equivalent forms, inventive embodiments can be by From be specifically described and require that different modes is put into practice.The inventive embodiments of present disclosure relates to described here Each independent feature, system, article, material, external member and/or method.If additionally, this category feature, System, article, material, external member and/or method are the most conflicting, two or more these category features, The combination in any of system, article, material, external member and/or method is included in the invention scope of present disclosure. Limit at this and dictionary definition that being defined of using is to be understood as outmatching limited term, lead to Cross the definition in the file quoted and combine and/or general sense.

All lists of references disclosed here, patents and patent applications are both relative to each theme being cited Combining by quoting, this can include the full content of file in some cases.

Unless clearly made contrary instruction, the most in the description and in the claims, as used herein Indefinite article "/kind) " (a/an) should be understood to be meant to " at least one (kind) ".

In the description and in the claims, phrase "and/or" as used herein should be understood to the meaning Refer to " any one or two " in the key element being so linked togather, i.e. key element is combined in some cases Ground exists and exists to separation property in other situations.The multiple elements listed by "and/or" should be with phase Same mode is explained, i.e. " one or more " in the element so combined.Except by "and/or" from The concrete element identified of sentence, other elements can be optionally present, either with concrete those identified unit Element is relevant or uncorrelated.Accordingly, as limiting examples, when combining open language such as " bag Include " use time, " A and/or B " is mentioned can the most only refer to A (optionally include except The element of B)；In another embodiment, B (optionally including the element except A) is only referred to；Again In one embodiment, refer to A and B (optionally including other elements)；Etc..

In the description and in the claims, "or" as used herein be interpreted as having with as more than The implication that defined "and/or" is identical.Such as, when separating the project in an inventory, "or" or " with/ Or " pardon should be construed to, i.e. include at least one in multiple element or element inventory, and Including therein more than one, and optionally, unlisted project additionally.The most clearly refer to The term shown, such as " only one in ... " or " in ... proper what a ", or want when be used for right When asking middle, " by ... composition " element just including in some or a column element will be referred to.Overall and Speech, term "or" as used herein ought above added with the term of exclusiveness, such as " any one ", " in ... one ", " ... middle only one " or " ... middle only one of which " time, it should it is interpreted only as instruction row The alternative form of his property (that is, " one or the other, but not both ")." mainly by ... composition " when with Time in claim, should have its its ordinary meaning as used by Patent Law field.

In the description and in the claims, as used herein, about having one or more key element The phrase " at least one " of inventory be interpreted as being meant in this key element inventory any one or multiple At least one key element of key element, but each key element being specifically set forth in being not necessarily included in this key element inventory In at least one, and be not excluded for any combination of multiple key elements in this key element inventory.This definition is also Allow, can be optionally present except specifically identifying in this phrase " at least one " indication element inventory Element outside element is either relevant or uncorrelated to those elements specifically identified.Therefore, As limiting examples, " at least one in A and B " (or equally, " at least in A or B Individual ", or equally " at least one in A and/or B ") may refer at least one in one embodiment Individual, optionally include more than one A, and there is not B (and optionally including the element except B)； In another embodiment, may refer at least one, optionally include more than one B, and there is not A (and optionally including the element except A)；In yet another embodiment, may refer at least one, Optionally include more than one A, and at least one, optionally include that more than one B is (and optionally Including other elements)；Etc..

It is also understood that contrary unless explicitly stated otherwise, include more than one step or action in this institute In claimed any method, the step of method or the order of action are not necessarily limited to the step of wherein method The order that rapid or action is listed.

In the claims, and in description above, all transitional phrases, such as " comprising ", " bag Include ", " carrying ", " having ", " containing ", " relating to ", " holding ", " by ... constitute " etc. be interpreted as open Property, i.e. it is meant to include but not limited to.Only transitional phrases " by ... composition " and " mainly By ... composition " should be respectively for closing or semi-closed transitional phrase, as USPO's patent is examined Look into procedure manual (United States Patent Office Manual of Patent Examining Procedures), described in Section 2111.03.

Claims

1. a method, including

(1) make one to need for the existence of one or more targets to carry out the sample tested and one Or the contact of multiple target specificity binding partners, the most each target specificity binding partners is connected to One butt chain, and wherein there is the most homospecific target specificity binding partners be connected to difference Docking core chain,

(2) unconjugated target specificity binding partners is optionally removed,

(3) the labeled imaging making this sample and there is the nucleotide sequence being complementary to a butt chain Chain contacts,

(4) unconjugated labeled imaging chain is optionally removed,

(6) quencher is from the signal of the labeled imaging chain of this combination, and

(7) repeat step (3)-(6), use one relative to all other labeled imaging chains every time For there is the labeled imaging chain of unique nucleotide sequence.

2. the method for claim 1, wherein this sample is special with more than one target in step (1) Specific binding partner contacts.

3. the method for claim 1, wherein this target specificity binding partners is a kind of antibody or anti- Body fragment.

4. the method for claim 1, wherein these labeled imaging chains are by labelling in the same manner.

5. the method for claim 1, the wherein each self-contained different mark of these labeled imaging chains Note.

6. the method for claim 1, wherein these labeled imaging chains are fluorescently-labeled imaging chains.

7. the method for claim 1, wherein these one or more targets are protein.

8. the method for claim 1, wherein this sample is that a kind of cell, cell lysate or tissue split Solve thing.

9. the method for claim 1, wherein uses copolymerization burnt or epifluorescence microscope inspection art is in step (5) by this sample imaging in.

10. the method for claim 1, wherein the quencher signal in step (6) includes photobleaching.

11. 1 kinds of compositionss, comprise:

The sample being attached on more than one target specificity binding partners, each combination Gametophyte is attached on a butt chain, and

At least one is stably coupled to the butt chain on a labeled imaging chain.

12. 1 kinds of methods, including

(1) make one to need for the existence of one or more targets to carry out the sample tested and one Or the contact of multiple target specificity binding partners, the most each target specificity binding partners is connected to One docking nucleic acid, and wherein there is the most homospecific target specificity binding partners be connected to not With docking nucleic acid,

(2) unconjugated target specificity binding partners is optionally removed,

(3) make this sample with have be complementary to one docking nucleic acid nucleotide sequence labeled become As nucleic acid contacts,

(4) unconjugated labeled imaging nucleic acid is optionally removed,

(6) by changing temperature and/or buffer condition by right from this for the labeled imaging nucleic acid of this combination Connect nucleic acid to remove, and

(7) repeat step (3)-(6), use one relative to all other labeled imaging cores every time There is for acid the labeled imaging nucleic acid of unique nucleotide sequence.

13. methods as claimed in claim 12, wherein this sample in step (1) with more than one target Specific binding partner contacts.

14. methods as claimed in claim 12, wherein this target specificity binding partners be a kind of antibody or Antibody fragment.

15. methods as claimed in claim 12, wherein this target specificity binding partners be a kind of natural or The part of through engineering approaches, little molecule, fit, peptide or oligonucleotide.

16. methods as claimed in claim 12, wherein these labeled imaging nucleic acid are by labelling in the same manner 's.

17. methods as claimed in claim 12, the wherein each self-contained difference of these labeled imaging nucleic acid Labelling.

18. methods as claimed in claim 12, wherein these labeled imaging nucleic acid are fluorescently-labeled imagings Nucleic acid.

19. methods as claimed in claim 12, wherein these one or more targets are protein.

20. methods as claimed in claim 12, wherein this sample is a kind of cell, cell lysate or tissue Lysate.

21. methods as claimed in claim 12, wherein use copolymerization burnt or epifluorescence microscope inspection art is in step Suddenly by this sample imaging in (5).

22. methods as claimed in claim 12, wherein these labeled imaging nucleic acid are dense by reducing salt Degree, interpolation denaturant or raising temperature are removed from these docking nucleic acid.

23. methods as claimed in claim 22, wherein this salt is Mg++.

24. methods as claimed in claim 22, wherein this denaturant is Methanamide, carbamide or DMSO.

25. methods as claimed in claim 12, wherein in the presence of a competition nucleic acid, are not put by chain Change commanders these labeled imaging nucleic acid from these docking nucleic acid removals.

26. methods as claimed in claim 12, wherein this unconjugated docking nucleic acid is partially double stranded.

27. methods as claimed in claim 12, wherein this unconjugated imaging nucleic acid is partially double stranded.

28. methods as claimed in claim 12, wherein this imaging nucleic acid is a molecular beacon or includes one Individual hairpin secondary structures.

29. methods as claimed in claim 12, wherein this imaging nucleic acid is a molecular beacon or includes one The hairpin secondary structures of individual self-quenching.

30. methods as claimed in claim 12, wherein this imaging nucleic acid is one and half duplexs.

31. methods as claimed in claim 30, wherein this half duplex is self-quenching.

32. methods as claimed in claim 12, wherein this docking nucleic acid includes a hairpin secondary structures.

33. methods as claimed in claim 12, wherein this imaging nucleic acid is by a dendritic structure or Individual paradigmatic structure is attached in multiple signal emission part.

34. 1 kinds of methods, including

(2) unconjugated target specificity binding partners is optionally removed,

(4) unconjugated labeled imaging nucleic acid is optionally removed,

(6) imaging nucleic acid is not exclusively removed by removing or modify their signal emission part Inactivate these labeled imaging nucleic acid combined, and

35. methods as claimed in claim 34, wherein this sample in step (1) with more than one target Specific binding partner contacts.

36. methods as claimed in claim 34, wherein this target specificity binding partners be a kind of antibody or Antibody fragment.

37. methods as claimed in claim 34, wherein this target specificity binding partners be a kind of natural or The part of through engineering approaches, little molecule, fit, peptide or oligonucleotide.

38. methods as claimed in claim 34, wherein these labeled imaging nucleic acid are by labelling in the same manner 's.

39. methods as claimed in claim 34, the wherein each self-contained difference of these labeled imaging nucleic acid Labelling.

40. methods as claimed in claim 34, wherein these labeled imaging nucleic acid are fluorescently-labeled imagings Nucleic acid.

41. methods as claimed in claim 34, wherein these one or more targets are protein.

42. methods as claimed in claim 34, wherein this sample is a kind of cell, cell lysate or tissue Lysate.

43. methods as claimed in claim 34, wherein use copolymerization burnt or epifluorescence microscope inspection art is in step Suddenly by this sample imaging in (5).

44. methods as claimed in claim 34, wherein this unconjugated docking nucleic acid is partially double stranded.

45. methods as claimed in claim 34, wherein this unconjugated imaging nucleic acid is partially double stranded.

46. methods as claimed in claim 34, wherein this imaging nucleic acid is a molecular beacon or includes one Individual hairpin secondary structures.

47. methods as claimed in claim 34, wherein this imaging nucleic acid is a molecular beacon or includes one The hairpin secondary structures of individual self-quenching.

48. methods as claimed in claim 34, wherein this imaging nucleic acid is one and half duplexs.

49. methods as claimed in claim 48, wherein this half duplex is self-quenching.

50. methods as claimed in claim 34, wherein this docking nucleic acid includes a hairpin secondary structures.

51. methods as claimed in claim 34, wherein this imaging nucleic acid is by a dendritic structure or Individual paradigmatic structure is attached in multiple signal emission part.